Therapeutic fusion proteins are, as a rule, artificial molecules, which are produced to combine known favorable properties of the single components or to create new properties. For example, a fusion protein may contain an immunogenic moiety that causes a normally non-immunogenic fusion partner to become immunogenic. In other cases, each of the components are immunogenic and the fusion molecule has kept this usually undesired property. Finally, it is possible that fusing non or less immunogenic components the fusion product is immunogenic by creating the bonds, especially the junction region.
Fusion proteins of specific interests in this context are immunoconjugates. Immunoconjugates are known since a couple of years and many of them have shown pharmacological efficacy in vitro and in vivo. Immunoconjugates are chimeric molecules consisting, as a rule, of a portion deriving from an immunoglobulin or a fragment thereof and a target polypeptide or protein which is linked to the immunoglobulin molecule. Originally, immunoconjugates were prepared consisting of a complete antibody and a cytotoxic agent like a cytokine which was fused via its N-terminus to the C-terminus of the constant domain of the immunoglobulin, or alternatively, via its C-terminus to the N-terminus of the variable region of the antibody (see, for example EP 0439 095, WO 92/08495, U.S. Pat. No. 5,349,053, EP 0659 439, EP 0706 799). These chimeric molecules are bi-functional by targeting a specific antigen, for example, on a tumor cell surface by means of the binding sites within the CDRs of the variable domain of the antibody portion or a fragment thereof, and by the simultaneous cytotoxic effect of the cytokine which is coupled to the immunoglobulin and thus can, theoretically, only or predominantly attack the targeted cell. In this context, also tri- and multi-functional immunoconjugates were developed including constructs consisting of sFv-, Fab-, Fab′ or F(ab′)2 fragments of different antibodies, wherein in each case the targeting function of the immunoglobulin portion was advantageously used.
Another immunoglobulin modification is the use of the Fc region of antibodies. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, “Fab”, “F(ab′)2”, dependent on the kind of digestion of the molecule, which bind antigen, and a constant domain, known as “Fc” which provides the link to effector functions such as complement or phagocytic cells. The Fc portion of an immunoglobulin has a long plasma half-life, whereas the Fab fragments are short-lived (Capon, et al., Nature 337: 525-531 (1989)).
Therapeutic protein products have been constructed using the Fc domain to provide longer half-life or to incorporate functions such as Fc receptor binding, protein A binding, complement fixation and placental transfer which all reside in the Fc proteins of immunoglobulins. For example, the Fc region of an IgG1 antibody has been fused to the N-terminal end of CD30-L, a molecule which binds CD30 receptors expressed on Hodgkin's Disease tumor cells, anaplastic lymphoma cells, T-cell leukemia cells and other malignant cell types (U.S. Pat. No. 5,480,981). IL-10, an anti-inflammatory and antirejection agent has been fused to murine Fcγ2a in order to increase the cytokine's short circulating half-life (Zheng et al., The Journal of Immunology, 154: 5590-5600 (1995)). Studies have also evaluated the use of tumor necrosis factor receptor linked with the Fc protein of human IgG1 to treat patients with septic shock (Fisher et al., N. Engl. J. Med., 334:1697-1702 (1996); Van Zee et al., The Journal of Immunology, 156:2221-2230 (1996)). Fc has also been fused with CD4 receptor to produce a therapeutic protein for treatment of AIDS (see: Capon et al., Nature, 337:525-531 (1989)). Principally, Fc can be fused to the target protein or peptide via its C- or N-terminus using the N- and C-terminus of the protein, respectively. A chimer of Fc and TNF and EPO was disclosed in EP 0464 533 (Hoechst/General Hospital), wherein the N-terminus of Fc was coupled to the C-terminus of the protein (X-Fc). The identical conjunction was selected for leptin-Fc chimers disclosed in WO 97/00319 (SKB) and WO 97/24440 (Genentech). There are a lot of publications and patent applications describing the opposite linkage of Fc-protein chimers (Fc-X), such as Fc-(IL-2), Fc-EPO, Fc-PSMA, Fc-(IL-12), Fc-TNFa, Fc-(GM-CSF), Fc-TNFR, Fc-endostatin, Fc, angiostatin, Fc-gp120, Fc-leptin, Fc-IFNa, Fc-(G-CSF). Examples are WO 96/08570, (Fuji/Merck KGaA), WO 98/28427 (Amgen), WO 99/02709 (Beth Israel Medical Care Center) and WO 99/58662 (Fuji/Merck KGaA). WO 00/24782 (Amgen) discloses a huge number of possible Fc-X conjugates, wherein the linkage between the two partners may be Fc-X or X-Fc. An extensive development of Fc-X molecules was realized by Lexigen/Merck KgaA as disclosed in U.S. Pat. No. 5,541,087, WO 99/43713, WO 99/29732, WO 99/52562, WO 99/53958, WO 00/11033, WO 01/07081, PCT/EP00/10843. Thus, X-Fc and Fc-X molecules which have “lost” their antigen binding sites, as well as molecules, wherein the binding sites und thus their antigen-specific targeting functions are conserved, are of great interest as promising therapeutic proteins and there exists a further need to develop analogue compositions for different clinical application.
Non-natural therapeutic proteins are often particularly immunogenic. For example, Enbrel is a fusion protein consisting of an extracellular domain of a Tumor Necrosis Factor Receptor (TNF-R) fused to an Fc region of an antibody. About 16% of patients treated with Enbrel have been reported to develop antibodies to this fusion protein (Physician's Desk Reference [2001] p. 3372). Similarly, a fusion protein consisting of erythropoietin (Epo) and granulocyte/macrophage-colony stimulating factor (GMCSF) was found to be highly immunogenic (Coscarella A, et al., [1998] Cytokine 10:964-9; Coscarella A, Mol Biotechnol. [1998] 10:115-22). When injected into a primate, Epo-GMCSF fusion proteins were found to induce a strong antibody response to the Epo moiety of the fusion protein, resulting in anemia. Ceredase™ and Cerezyme™ are forms of the lysosomal enzyme glucocerebrosidase used to treat Gaucher's disease; as a result of genetic engineering, glucocerebrosidase is attached to an unusual high-mannose glycosylation. Patients with Gaucher's disease lack glucocerebrosidase in their lysosomes, and as a result the patients' macrophages tend to accumulate lipids and become foam cells (The Metabolic and Molecular Bases of Inherited Disease, 8th Edition [2001] Scriver et al., eds., Chapter 146, “Gaucher Disease,” p. 3635-3668). After administration of Ceredase™ or Cerezyme™, the therapeutic protein is bound by mannose receptors on macrophages, endocytosed, and trafficked through the endosomes to the lysosome, which is its proper location. Patients treated with Ceredase often develop antibodies to glucocerebrosidase (Pastores G M, et al., Blood [1993] 82:408-16; Physicians' Desk Reference [2001] p. 1325-1326). Such antibodies can interfere with treatment (Brady R O, et al., Pediatrics [1997] 100(6):E11.). In a Phase I clinical trial using an antibody-cytokine fusion protein, several patients developed antibody responses to the therapeutic fusion protein. In this case, the antibody moiety was a humanized form of the 14.18 antibody, and the cytokine was interleukin-2 (IL-2). Many of the reactive patients' sera included significant levels of anti-idiotype antibodies.
Therapeutic use of a number of peptides, polypeptides and proteins is curtailed because of their immunogenicity in mammals, especially humans. For example, when murine antibodies are administered to patients who are not immunosuppressed, a majority of such patients exhibit an immune reaction to the introduced foreign material by making human anti-murine antibodies (HAMA) (e.g. Schroff, R. W. et al., Cancer Res. 45:879-885 (1985); Shawler, D. L. et al., J. Immunol. 135:1530-1535 (1985)). There are two serious consequences. First, the patient's anti-murine antibody may bind and clear the therapeutic antibody or immunoconjugate before it has a chance to bind, for example to a tumor, and perform its therapeutic function. Second, the patient may develop an allergic sensitivity to the murine antibody and be at risk of anaphylactic shock upon any future exposure to murine immunoglobulin.
Several techniques have been employed to address the HAMA problem and thus enable the use in humans of therapeutic monoclonal antibodies (see, for example, WO89/09622, EP0239400, EP0438310, WO91/09967). These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant “humanized” antibodies have, in several cases, still elicited an immune response in patients (Issacs, J. D., Sem. Immunol. 2:449, 456 (1990); Rebello, P. R. et al., Transplantation 68:1417-1420 (1999)).
A common aspect of these methodologies has been the introduction into the therapeutic antibody, usually of rodent origin, of amino acid residues, even significant tracts of amino acid residue sequences, identical to those present in human antibody proteins. For antibodies, this process is possible owing to the relatively high degree of structural (and functional) conservatism among antibody molecules of different species. For potentially therapeutic peptides, polypeptides and proteins, however, where no structural homologue may exist in the host species (e.g., human) for the therapeutic protein, such processes are not applicable.
Furthermore, these methods have assumed that the general introduction of a human amino acid residue sequence will render the re-modeled antibody non-immunogenic. It is known, however, that certain short peptide sequences (“T-cell epitopes”) can be released during the degradation of peptides, polypeptides or proteins within cells and subsequently be presented by molecules of the major histocompatability complex (MHC) in order to trigger the activation of T-cells. For peptides presented by MHC Class II, such activation of T-cells can then give rise to an antibody response by direct stimulation of B-cells to produce such antibodies. Accordingly, it would be desirable to eliminate potential T-cell epitopes from a peptide, polypeptide or a protein. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans. Notable examples include therapeutic use of granulocyte-macrophage colony stimulating factor (Wadhwa, M. et al., Clin. Cancer Res. 5:1353-1361 (1999)) and interferon alpha 2 (Russo, D. et al., Bri. J. Haem. 94:300-305 (1996); Stein, R. et al., New Engl. J. Med. 318:1409-1413 (1988)).
During the last couple of years several techniques were published which suggest solutions for rendering antibodies and target proteins having different biological functions non- or at least less immunogenic. Examples are: WO 92/10755 and WO 96/40792 (Novo Nordisk), EP 0519 596 (Merck & Co.), EP 0699 755(Centro de Immunologia Moelcular), WO 98/52976 and WO 98/59244 and WO 00/34317 (Biovation Ltd.).
The general methods disclosed in the prior art and regarding the elimination of T-cell epitopes from proteins (e.g. WO 98/52976, WO 00/34317) comprise the following steps:    (a) Determining the amino acid sequence of the polypeptide or part thereof    (b) Identifying one or more potential T-cell epitopes within the amino acid sequence of the protein by any method including determination of the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays.    (c) Designing new sequence variants with one or more amino acids within the identified potential T-cell epitopes modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope as determined by the binding of the peptides to MHC molecules using in vitro or in silico techniques or biological assays. Such sequence variants are created in such a way to avoid creation of new potential T-cell epitopes by the sequence variations unless such new potential T-cell epitopes are, in turn, modified in such a way to substantially reduce or eliminate the activity of the T-cell epitope.    (d) Constructing such sequence variants by recombinant DNA techniques and testing said variants in order to identify one or more variants with desirable properties.
Other techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides and able to bind to T-cell clones from peripheral blood samples from human or experimental animal subjects have been used in the art [Kern, F. et al., Nature Medicine 4:975-978 (1998); Kwok, W. W. et al., TRENDS in Immunology 22:583-588 (2001)] and may also be exploited in an epitope identification strategy.
The potential T-cell epitopes are generally defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Such potential T-cell epitopes can be measured to establish MHC binding. In the general understanding the term “T-cell epitope” is an epitope which when bound to MHC molecules can be recognized by the T-cell receptor, and which can, at least in principle, cause the activation of these T-cells. It is, however, usually understood that certain peptides which are found to bind to MHC Class II molecules may be retained in a protein sequence because such peptides are tolerated by the immune within the organism into which the final protein is administered.
The invention is conceived to overcome the practical reality that soluble proteins introduced into an autologous host with therapeutic intent, can trigger an immune response resulting in development of host antibodies that bind to the soluble protein. One example amongst others is interferon alpha 2 to which a proportion of human patients make antibodies despite the fact that this protein is produced endogenously [Russo, D. et al., Brit. J. Haem. 94:300-305 (1996); Stein, R. et al., New Engl. J. Med. 318:1409-1413 (1988)].
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins and the major focus of the present invention. However, isotypes HLA-DQ and HLA-DP perform similar functions, hence the present invention is equally applicable to these. MHC HLA-DR molecules are homo-dimers where each “half” is a hetero-dimer consisting of α and β chains. Each hetero-dimer possesses a ligand binding domain which binds to peptides varying between 9 and 20 amino acids in length, although the binding groove can accommodate a maximum of 9-11 amino acids. The ligand binding domain is comprised of amino acids 1 to 85 of the α chain, and amino acids 1 to 94 of the β chain. DQ molecules have recently been shown to have an homologous structure and the DP family proteins are also expected to be very similar. In humans approximately 70 different allotypes of the DR isotype are known, for DQ there are 30 different allotypes and for DP 47 different allotypes are known. Each individual bears two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and such structures point to an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide [Brown et al., Nature 364:33 (1993); Stem et al., Nature 368:215 (1994)].
Polymorphism identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
There is a considerable amount of polymorphism within the ligand binding domain with distinct “families” within different geographical populations and ethnic groups. This polymorphism affects the binding characteristics of the peptide binding domain, thus different “families” of DR molecules will have specificities for peptides with different sequence properties, although there may be some overlap. This specificity determines recognition of Th-cell epitopes (Class II T-cell response) which are ultimately responsible for driving the antibody response to B-cell epitopes present on the same protein from which the Th-cell epitope is derived. Thus, the immune response to a protein in an individual is heavily influenced by T-cell epitope recognition which is a function of the peptide binding specificity of that individual's HLA-DR allotype. Therefore, in order to identify T-cell epitopes within a protein or peptide in the context of a global population, it is desirable to consider the binding properties of as diverse a set of HLA-DR allotypes as possible, thus covering as high a percentage of the world population as possible.
A principal factor in the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cell via presentation on MHC class II molecules. In order to eliminate or reduce immunogenicity, it is thus desirable to identify and remove T-cell epitopes from the protein.
According to the above-cited methods and related processes several biological molecules, basically usual target proteins and antibodies have been prepared which reveal reduced immunogenicity and allergenicity. Examples are: WO 99/55369 (SKB), WO 99/40198 and WO 96/21016 (Leuven Research & Development VZW), WO 00/08196 (Duke University), WO 96/21036 (Chiron Viragen), WO 97/31025 (Chiron Corp.), WO 98/30706 (Alliance Pharmaceutical Corp.).
In all these applications cited above single proteins or antibodies eliciting a lower immune response were disclosed; there is no hint that fusion proteins, above all immunoglobulin fusion proteins were completely or partially de-immunized, especially by reducing the number of T-cell epitopes within the sequence of said molecules by means of partially computational methods. In WO 97/24137 (Tannox Biosystems Inc.) a IFNα-Fc chimer is disclosed which contains a non-immunogenic linker molecule between the N-terminus of the Fc portion and the C-terminus of IFNα.
Therefore, it is still a need to provide for biological molecules, such as immunoconjugates, which are not or less immunogenic. Above all, it is of specific interest to provide for Fc-conjugates, preferably Fc-X chimers, wherein X is a selected protein or polypeptide of therapeutic interest.